文章摘要
Hongyu Yang, Hong Qi,Junjie Zou,Xi-wei Zhang.[J].南京医科大学学报,2008,28(1):52~56
Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor
投稿时间:2007-11-23  
DOI:10.7655
中文关键词: 
英文关键词: vascular endothelial growth factor  adenovirus vector  genetic transfection
基金项目:
作者单位
Hongyu Yang Department of Vascular Surgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029,Jiangsu Province, China 
Hong Qi School of Public Health, Nanjing Medical University, Nanjing 210029,Jiangsu Province, China 
Junjie Zou Department of Vascular Surgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029,Jiangsu Province, China 
Xi-wei Zhang Department of Vascular Surgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029,Jiangsu Province, China 
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中文摘要:
      
英文摘要:
      Objective: To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF), as preparation for genetic transfection that follows. Methods:Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by Lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF and VDC316-VEGF was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose(TCID50) assay. Results:The newly constructed recombinant adenovirus was confirmed to carry rat VEGF based on PCR results, and its titration value determined based on TCID50 assay was 3×109 pfu/ml. Conclusion:The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a sufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.
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